In recent years it has been widely recognized that the environment has been contaminated with toxic substances, or toxicants, of natural and industrial origins which can, even in trace amounts, cause toxic effects on humans, domesticated animals, and fish and wildlife. Such toxicants can be present in trace amounts in air, soil, water, or foods and foodstuffs and can produce acute or chronic adverse somatic effects in humans or animals exposed to or ingesting these materials, as well as mutagenic, teratogenic or carcinogenic effects. It has therefore become a requirement of our society that environmental sources of substances essential to life, such as surface or ground water used as sources of drinking water, must be examined for trace amounts of an array of toxic substances such as heavy metals, chlorinated pesticides, and volatile organic industrial chemicals, which are very difficult to detect, yet which even in trace amounts may exert deleterious affects on humans or domesticated animals consuming the water. Similar concerns also apply to the protection of fish and wildlife which live in or near lakes and streams which have been inadvertently polluted by man with similar toxicants resulting from human industry. Some of the toxicants, even though present at very low levels in aquatic water systems, are concentrated by biological activity, such as occurs with some chlorinated pesticides and polychlorinated biphenyls and other similar compounds, to thus cause deleterious affects on higher life forms, including both wildlife and humans which consume fish from contaminated waters.
It is therefore useful to have assays which can detect the presence of toxic substances in environmental samples at very low levels. Ideally, it would be beneficial if such assays could be qualitative as well as quantitative by indicating the type of chemical substance which is detected if a positive result is obtained from the assay. However, both qualitative and accurate guantitative chemical analyses of an unknown sample of toxicant are at present very slow and expensive processes. Mixtures present special difficulties because of the need to conduct separate analyses for the constituents thereof. Sometimes even the appropriate analytical methodology and/or toxicological information is not available for individual compounds, particularly new ones. The situation can be even further complicated when multiple toxicants are present in single environmental samples since the interaction between the toxicants can result in additive, synergistic or antagonistic interaction with results that are difficult to predict. Nevertheless, even if qualitative analysis of samples is impractical, sensitive quantitative analysis of samples to screen for the presence of deleterious, even if unknown, toxicants is of great use in determining the safety or the level of contamination of environmental areas. To provide effective tools in risk assessment and to attempt to cope with the problems of rapid assessment of the toxicity of environmental resources, scientists and regulatory officials have sought for some time to develop suitable bioassays for use in quickly screening or examining environmental samples which can be both economical and sensitive. Bioassys typically measure the response of a biological preparation or whole organism to toxic challenges from a sample of unknown constituents, without the need for identifying or quantifying the chemicals concerned, other than quantitatively measuring their effect on biological activity. It has been found that data from such bioassay tests correlate well with affects on laboratory animals and humans when determined by conventional toxicological or epidemiological data. Some prior bioassays have been developed which are based on relatively simple biochemical tests, such as those based on single enzymes or groups of enzymes. Another type of prior bioassay is one based on the responses of whole organisms. One example of a test based on a whole organism approach is the well-known Ames tests for mutagenicity which tests the effect of a sample of known or unknown character on the rate of mutagenic effect on a bacterial strain of known genetic character. Typical bioassays to test unknown environmental toxicants have been done with whole higher organisms such as Cereodaphnia magna, water fleas, or selected fish species, such as minnows or rainbow trout, which are sensitive to toxicological effects. One previously known bioassay using whole organisms, in this case bacteria, is based on the light output of the bioluminescent bacterium Photobacterium phosphoreum and is marketed by Beckman Instruments under the trade name Microtox. The Mictrotox test utilizes the bioluminescent bacteria, and measures their sensitivity to toxic substances, by noting the quenching of the bioluminescence as the bacteria are adversely affected by dosages of the toxicant.
There is still a need for efficient and economical assays. There is feeling that simple in vitro tests, as for example those using enzymes, are too far removed from whole organisms to qualify as good surrogates, since they are often not sensitive enough to a wide spectrum of toxic agents. On the other end, whole organism tests are often time-consuming and expensive. There are additional problems with whole organism tests, particularly those dependent on microorganisms, in that mutations may cause biological variability in the microorganisms which can cause the results to vary, and in that bacterial cell walls may inhibit the uptake of toxicants, producing falsely negative results in tests.